Figure 6.

SHP-1 Can Replace SHP-2 for PD-1 Coinhibition in Jurkat T Cells

(A) Immunoblot analysis of equal amounts of proteins from total lysates of Raji (WT), Raji PD-L1, Raji SHP-2^–, and Raji PD-L1 SHP-2^– cells probed with antibodies to SHP-1 (right panel), SHP-2 (left panel), or VAV1 (loading control). Left margin, molecular size in kilodaltons. Data are representative of two independent experiments.

(B) Raji, Raji PD-L1, Raji SHP-2^–, and Raji PD-L1 SHP-2^– cells were analyzed using flow cytometry for expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM. Gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.

(C) Jurkat-PD-1^OST and Jurkat-PD-1^OST SHP-2^– cells were stimulated with Raji SHP-2^– cells or Raji PD-L1 SHP-2^– cells that have been preincubated in the absence (–) or presence (+) of SEE and lysed for 2 min after the initial contact. Immunoblot analysis of equal amounts (90%) of lysates from the specified conditions subjected to affinity purification (AP) on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin, and probed with antibody to anti-SHP-1, anti-SHP-2, and phosphorylated proteins (Anti-p-Tyr). Also shown is immunoblot analysis of equal amounts (10%) of total lysates of the specified cells probed with anti-PD-1 antibody (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent experiments.